Surface receptors for peanut agglutinin (PNA), a lectin with D-galactose specificity, were detected on mouse thymocytes using fluorescence microscopy. Depending on mouse strain, 69-85% of unseparated thymocytes could thus be characterized as PNA+. Electrophoretic fractionation of thymocytes from normal or immunosuppressive drug-treated donors revealed an inverse relationship between PNA-binding properties and cell electrophoretic mobility (EPM). Thus, all thymocytes recovered in the lowest EPM fractions were strongly PNA+ whereas those in the highest EPM fractions were in the majority PNA-. Most of the cells collected in the intermediate EPM range were PNA+ but staining with the fluoresceinated lectin appeared weaker than for the low EPM thymocytes. Reciprocal experiments in which thymocytes were separated by PNA-mediated aggregation into fractions with different affinities for the lectin and then subjected to physical analysis, definitely established that PNA+ cells are of lower EPM than PNA- cells and that these two cell types also differ in size distribution. These data show that the four physical subpopulations of thymocytes previously described present distinctive PNA-binding properties: Th1 and Th2 cells can be classified as strongly PNA+, Th3 cells as less intensely PNA+, and Th4 cells as mostly PNA-.